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author:("bilis, nyhan")
1.  Notch signaling pathway in cumulus cells can be a novel marker to identify poor and normal responder IVF patients 
To identify expression of Notch signaling proteins and its ligands in human cumulus cells which were obtained by follicle aspiration and to compare the differences of this protein expression between the normal and poor responder patients.
47 patients who applied to the assisted reproductive treatments with various infertility problems were included to the study. Controlled ovarian hyperstimulation was performed by using GnRH agonist and gonadotropins. Serum hormon levels were measured by using Chemilluminescent Microparticle Immunoassay method for each patient. After ultrasonographic ovarian follicle screening, oocytes were retrievaled. Cumulus cells obtained from the follicles were cultured for 72 h and immunuhistochemistry were performed for Notch1, Notch2, Notch3, Notch4, Jagged1 and Jagged2 proteins. Histological score (HSCORE) were applied to all of the samples. The association between Notch and its ligands protein expressions and the oocyte-embryo quality and fertilization rates were investigated.
Significant differences were observed between the mean values of age, AMH and FSH in the 2 groups, respectively (p < 0.05). However, the mean female infertility duration and total gonadotropin dose did not differ significantly between normal and poor responder groups. All the patients cumulus cells expressed Notch1, Notch2, Notch3, Notch4, Jagged1 and Jagged2. There was a significant difference (p < 0.05) only for Notch2 between the 2 groups and a positive correlation between Notch2 and Notch3 (r = 547, p = 0.00) expressions were noted. Furthermore, no correlations were observed between the following: Notch1, Notch2, Notch3, Notch4, Jagged1, and Jagged2 expression; mature oocyte number; fertilization rates, and embryo quality percentage in both of the groups.
Notch signalling proteins can be an indicator for understanding the ovarian response in ovulation induction.
PMCID: PMC3824856  PMID: 23922013
Notch signalling; Cumulus cells; Poor responder; Ovary follicles; In vitro fertilization
2.  Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells 
Oncology Letters  2014;7(6):2103-2109.
Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.
PMCID: PMC4049671  PMID: 24932297
cancer stem cell; stem cell-related genes; spheroid; prostate cancer
3.  Combination of imatinib mesylate with lithium chloride and medroxyprogesterone acetate is highly active in Ishikawa endometrial carcinoma in vitro 
Journal of Gynecologic Oncology  2011;22(4):225-232.
The aim of the study was to investigate whether lithium chloride and medroxyprogesterone acetate can potentiate the cytotoxicity of imatinib mesylate in human endometrial cancer in vitro and the effect of midkine in these therapies.
Imatinib mesylate (50 µM), lithium chloride (100 µM), medroxyprogesterone acetate (200 µM) and their combination were applied to monolayer and three dimensional cultures of human Ishikawa endometrial cancer for 72 hours. The cell proliferation index, apoptotic index, caspase-3 and midkine levels, cell cycle distributions in monolayer cultures and cell ultrastructure in spheroid cultures were evaluated. Results were statistically analyzed using the Student's t-test.
All drug applications inhibited cell proliferation (p<0.05), however the combination were the effective groups for 72 hours (p<0.05). Interestingly, although the loss of efficiency was seen higly seen every 24 hours at single applications, the inhibition rates of the combination groups were almost same for 72 hours. In concordance with these results, the apoptotic index, caspase-3 levels (p<0.05), cell morphology and ultrastructure damages were much higher in the combination groups. Imatinib mesylate induced S-phase arrest, however other groups induced G0+G1-phase arrest at 24 hours and all groups induced G0+G1 arrest at 72 hours (p<0.05). Imatinib mesylate and imatinib mesylate with medroxyprogesterone acetate induced highest decrease in midkine levels, respectively (p<0.05).
The present study showed that the combination of imatinib mesylate with lithium chloride and medroxyprogesterone acetate is highly active in Ishikawa endometrial carcinoma in vitro and the inhibition of midkine involved in their mechanism of action against endometrium defense.
PMCID: PMC3254840  PMID: 22247798
Endometrial cancer; Imatinib mesylate; Lithium chloride; Medroxyprogesterone acetate; Midkine
4.  Imatinib mesylate decreases the cytotoxic effect of roscovitine on human glioblastoma cells in vitro and the role of midkine 
Oncology Letters  2011;3(1):200-208.
The purpose of the present study was to overcome resistance to imatinib (IM) by combining it with roscovitine (ROSC) and to investigate whether or not midkine (MK) had an effect on this combination in the treatment of glioblastoma (GBL). Human T98 GBL cells were used to evaluate the effects of IM (10 μM), ROSC (200 μM) and their combination on the cell proliferation index, apoptotic index, the apoptotic protein and anti-apoptotic protein levels, and ultrastructure. All applications decreased the cell proliferation index and increased the apoptotic index, but ROSC was the most efficient drug and the second most efficient drug was IM. Notably, ROSC increased anti-apoptotic proteins levels (PDGFR-α, AQP-4, hTERT), COX-1 activity and ribosome numbers. The effects of ROSC on hTERT, MK, AQP-4 and MRP-1 levels and COX-1 activity were reported for the first time. ROSC induced the highest increase in caspase-3 levels. Autophagy was not involved in the activity of ROSC in GBL spheroids. The combination of IM with ROSC showed an antagonist effect in the treatment of human GBL cells. The combination group decreased certain anti-apoptotic protein levels (PDGFR-α, EGFR, p-gp, MRP-1 and MK), cAMP levels, COX-1 activity and apoptotic protein levels (caspase-3). However, it induced the highest increase in hTERT levels and COX-2 activity. Ribosome numbers were much lower than those in the ROSC group and no autophagic vacuole was observed. In conclusion, more investigations are required to identify the key regulatory components that are responsible for this antagonism; however, the determination of this combination therapy as a failure therapy may be precautionary for oncologists in the treatment of GBL patients and potentially may contribute to the efficacy of new therapeutic regimens.
PMCID: PMC3362508  PMID: 22740881
imatinib mesylate; roscovitine; glioblastoma; midkine; combination chemotherapy
5.  Decreased therapeutic effects of noscapine combined with imatinib mesylate on human glioblastoma in vitro and the effect of midkine 
Glioblastoma (GBM) develops resistance to the advances in chemotherapy leading to poor prognosis and life quality. Consequently, new treatment modalities are needed. Our aims were to investigate the effects of combined noscapine (NOS) and imatinib mesylate (IM) on human GBM in vitro and the role of midkine (MK) in this new combination treatment.
Monolayer and spheroid cultures of T98G human GBM cell line were used to evaluate the effects of IM (10 μM), Nos (10 μM) and their combination on cell proliferation and apoptotic indexes, cell cycle, the levels of antiapoptotic MK, MRP-1, p170, PFGFR-α, EGFR, bcl-2 proteins, apoptotic caspase-3 levels, morphology (SEM) and ultrastructure (TEM) for 72 hrs. Results were statistically analyzed using the Student's t-test.
The combination group induced highest decrease in cell proliferation and apoptotic indexes, caspase-3 levels, MRP-1 and PDGFR-α levels. The decrease in p170 levels were lower than IM but higher that NOS. The highest increases were in EGFR, MK, bcl-2 and cAMP levels in the combination group. The G0+G1 cell cycle arrest at the end of 72nd hr was the lowest in the combination group. Apoptotic appearence was observed rarely both in the morphologic and ultrastructural evaluation of the combination group. In addition, autophagic vacuoles which were frequently observed in the IM group were observed rarely.
The combination of Nos with IM showed antagonist effect in T98G human GBM cells in vitro. This antagonist effect was correlated highly with MK levels. The effects of NOS on MRP-1, MK and receptor tyrosine kinase levels were firstly demonstrated in our report. In addition, we proposed that MK is one of the modulator in the switch of autophagy to cell death or survival/resistance.
PMCID: PMC3135492  PMID: 21651812
6.  Evaluation of Light-Emitting Diode (LED-660 Nm) Application over Primary Osteoblast-Like Cells on Titanium Surfaces: An In Vitro Study 
Background: The goal of this study was to evaluate the behavior of neonatal rat calvarial osteoblast-like cells cultured on different implant surfaces and exposed once or three times to a 660-nm light-emitting diode (LED).
Methods: An LED with a 660-nm wavelength was applied once or three times to cultured cells on standard and modified sandblasted acid-etched surfaces (SLA and SLActive; Straumann, Basel, Switzerland). To analyze the effect of the LED on cell proliferation, numbers, and viability, cells were cultured on titanium discs, and measurements were taken after 72 h. Cell proliferation rates were assessed using a bromodeoxyuridine immunohistochemical technique. Cell morphologies were evaluated by scanning electron microscopy (SEM).
Results: Osteoblast-like cells proliferated on all tested surfaces, with differences among groups in cell counts and DNA synthesis values. The application of one LED treatment caused a significant increase in cell count in the SLActive group in comparison with the SLA group (p = 0.001), whereas the application of three LED treatments caused a significant decrease in cell count in the SLA group compared with the SLActive group (p < 0.001). After 72 h, the number of cells was highest in the SLActive group exposed once to the LED.
Conclusions: One LED application in the SLActive group resulted in significantly increased cell numbers. However, these findings were not exactly compatible with the SEM findings, which demonstrated fewer cells and weak attachments between cells and to the surface. Thus, further studies using different LED application times are needed to clarify the reason for the increased number of cells that are apparently incapable of attaching to the titanium surfaces after 72 h.
PMCID: PMC3198254  PMID: 22022211
Light-emitting diode; SLA and SLActive surface; primary osteoblast cell culture
7.  Is the thymidine labeling index a good prognostic marker in breast cancer? 
The aim of the present study was to determine the prognostic relevance of thymidine labeling index (TLI) in patients with breast cancer.
TLI of the primary tumor was measured in 268 patients at the time of the surgical biopsy by an in vitro method.
Fifty-four patients had stage I disease, and 138 patients had stage II disease, and 76 patients had stage III disease. One hundred-four patients were found to have low TLI-index (<3%), and 164 patients had high TLI-index (≥3%). The median follow-up was 71.5 months (range, 6–138 months). The 5-year overall survival (OS) and disease free survival (DFS) rates was 84% and 74%, respectively. Lymph node involvement, tumor size more than 2 cm, high nuclear grade and estrogen receptor negativity were found to be associated with poorer DFS and OS rates. On subgroup analysis, however, the 5-year OS rate was significantly higher in the low TLI-group than in the high TLI-group in patients with stage I disease (100% vs 76%, p = 0.05).
Our findings suggest that the prognostic significance of TLI appears to be limited to early breast cancer that might help to distinguish patients who need more aggressive adjuvant treatment.
PMCID: PMC2000894  PMID: 17705874
8.  Suramin Increased Telomerase Activity in the C6 Glioma/Wistar Experimental Brain Tumor Model 
Glioblastoma multiforme (GBM) is the most treatment-resistant glioma variant. Significant roles for telomerase in etiology, recurrence and drug resistance of GBM have been highlighted. Suramin (Bayer, Leverkusen, Germany) is an antineoplastic agent that affects many cellular mechanisms including growth factor, purinergic receptor, cytokine and key cellular enzymes signaling. The aim of this study was to investigate whether suramin, 40 mg/kg, i.p., inhibits telomerase activity in a subcutaneous C6 glioma/Wistar experimental brain tumor model using PCR based telomeric repeat amplification assay. In comparison to the control group, suramin increased tumor volume and telomerase activity. We also used transmission electron microscopy to evaluate the alterations of cell morphology. Apoptosis was seen markedly in electron micrographs of the control group and anti-apoptotic activity of telomerase was verified in the electron micrographs of suramin-applied group. The in vitro inhibitory effects of suramin on telomerase activity in several cell lines except for brain tumors have been reported. Contrary to in vitro reports, our results were the first to demonstrate that suramin increased telomerase activity in a C6 glioma/Wistar experimental brain tumor. Large numbers of drugs exhibited apparent hormetic effects on cultured cancer cells and in vivo cancer growth. Several drug examples for their hormetic effects in vivo were listed as resveratrol, suramin, and tamoxifen. The action of suramin in the present study could be evaluated as one of the hormetic examples of suramin in vivo.
PMCID: PMC3614628  PMID: 23675031
chemosensitivity; C6 glioma; hormesis; suramin; telomerase

Results 1-8 (8)